title: Membrane damage by an α-helical pore-forming protein, Equinatoxin II, proceeds through a succession of ordered steps.
abstract: Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Nejc
authors.initials: N
authors.lastname: Rojko
authors.forename: Katarina Č
authors.initials: KČ
authors.lastname: Kristan
authors.forename: Gabriella
authors.initials: G
authors.lastname: Viero
authors.forename: Eva
authors.initials: E
authors.lastname: Žerovnik
authors.forename: Peter
authors.initials: P
authors.lastname: Maček
authors.forename: Mauro
authors.initials: M
authors.lastname: Dalla Serra
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000494:pharmacology*; D003545:Cysteine / Q000235:genetics; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008563:Membrane Lipids / Q000737:chemistry; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D055503:Protein Multimerization / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
chemical_list: D003064:Cnidarian Venoms; D008563:Membrane Lipids; D050505:Mutant Proteins; C012307:equinatoxin; D003545:Cysteine
keywords: Actinoporin; Equinatoxin; Erythrocyte; Fluorescence; Kinetics; Membrane; Pore Forming; Toxins